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BioAssay: AID 651947

HepG2 Cytotoxicity Assay Measured in Cell-Based System Using Plate Reader - 7071-02_Other_Dose_DryPowder_Activity

Assay Overview: The Counter Screen use wild-type HepG2 cells from ATCC to counter screen for cytotoxicity at the same concentrations used to confirm compound activity in the primary screen. ..more
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 Tested Compounds
 Tested Compounds
All(132)
 
 
Active(12)
 
 
Inactive(111)
 
 
Inconclusive(9)
 
 
 Tested Substances
 Tested Substances
All(133)
 
 
Active(12)
 
 
Inactive(112)
 
 
Inconclusive(9)
 
 
 Related BioAssays
 Related BioAssays
AID: 651947
Data Source: Broad Institute (7071-02_Other_Dose_DryPowder_Activity)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
BioAssay Version:
Deposit Date: 2012-12-16
Hold-until Date: 2013-09-07
Modify Date: 2013-09-07

Data Table ( Complete ):           View Active Data    View All Data
BioActive Compounds: 12
Related Experiments
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AIDNameTypeProbeComment
488929Elucidation of physiology of non-replicating, drug-tolerant Mycobacterium tuberculosis - SummarySummary1 depositor-specified cross reference: Summary
687034Broad Institute Elucidation of the physiology of non-replicating, drug tolerant Mycobacterium tuberculosis: Probe to inhibit the Transition from Non-Replication to Replication Inhibitor Probe ProjectSummary depositor-specified cross reference
488890Elucidation of physiology of non-replicating, drug-tolerant Mycobacterium tuberculosisConfirmatory same project related to Summary assay
489018A Cell Based Secondary Assay to Explore Cytotoxicity in HepG2 Cells of Compounds that Modulate Non-Replicating, Drug-tolerant Mycobacterium tuberculosisConfirmatory same project related to Summary assay
489025A Cell Based Secondary Assay to Explore Cytotoxicity in THP-1 Cells of Compounds that Modulate Non-Replicating, Drug-tolerant Mycobacterium tuberculosisConfirmatory same project related to Summary assay
492952A Cell Based Secondary Assay to Explore Compounds that Modulate Non-Replicating, Drug-tolerant Compounds in Replicating H37Rv TB of Mycobacterium tuberculosisConfirmatory same project related to Summary assay
492998A Cell Based Secondary Assay to Explore Cytotoxicity in Vero E6 Cells of Compounds that Modulate Non-Replicating, Drug-tolerant Mycobacterium tuberculosisConfirmatory same project related to Summary assay
651617Counterscreen for inhibitors of non replicating M. tb using log phase replicating mycobacteria Measured in Microorganism System Using Plate Reader - 2157-02_Inhibitor_Dose_DryPowder_ActivityConfirmatory same project related to Summary assay
651618Counterscreen for inhibitors of non replicating M. tb using log phase replicating mycobacteria Measured in Microorganism System Using Plate Reader - 2157-02_Inhibitor_Dose_DryPowder_Activity_Set2Confirmatory same project related to Summary assay
651619Secondary assay to identify inhibitors of non-replicating M. tb using luciferase expression without log phase outgrowth Measured in Microorganism System Using Plate Reader - 2157-04_Inhibitor_Dose_DryPowder_Activity_Set2Confirmatory same project related to Summary assay
651620Secondary assay to identify inhibitors of non-replicating M. tb using luciferase expression without log phase outgrowth Measured in Microorganism System Using Plate Reader - 2157-04_Inhibitor_Dose_DryPowder_ActivityConfirmatory same project related to Summary assay
651850Inhibition of Mycobacterium tuberculosis during logarithmic growth, as enumerated by colony forming units_2157-05_Inhibitor_Dose_DryPowder_ActivityConfirmatory same project related to Summary assay
651851Counterscreen for inhibitors of non replicating M. tb using log phase replicating mycobacteriaConfirmatory same project related to Summary assay
651856Secondary assay to identify inhibitors of non-replicating M. tb using luciferase expression without log phase outgrowthConfirmatory same project related to Summary assay
651857Inhibition of Mycobacterium tuberculosis during starvation growth, as enumerated by colony forming units_2157-06_Inhibitor_Dose_DryPowder_ActivityConfirmatory same project related to Summary assay
651946HeLa Cytotoxicity Assay Measured in Cell-Based System Using Plate Reader - 7071-04_Other_Dose_DryPowder_ActivityConfirmatory same project related to Summary assay
651949HEK293 Cytotoxicity Assay Measured in Cell-Based System Using Plate Reader - 7071-01_Other_Dose_DryPowder_Activity_Set6Confirmatory same project related to Summary assay
687033Broad Institute Elucidation of the physiology of non-replicating, drug tolerant Mycobacterium tuberculosis: Probe for both Non-Replicating & Replicating organisms Inhibitor Probe ProjectSummary same project related to Summary assay
488890Elucidation of physiology of non-replicating, drug-tolerant Mycobacterium tuberculosisConfirmatory same project related to Summary assay
489018A Cell Based Secondary Assay to Explore Cytotoxicity in HepG2 Cells of Compounds that Modulate Non-Replicating, Drug-tolerant Mycobacterium tuberculosisConfirmatory same project related to Summary assay
489025A Cell Based Secondary Assay to Explore Cytotoxicity in THP-1 Cells of Compounds that Modulate Non-Replicating, Drug-tolerant Mycobacterium tuberculosisConfirmatory same project related to Summary assay
492952A Cell Based Secondary Assay to Explore Compounds that Modulate Non-Replicating, Drug-tolerant Compounds in Replicating H37Rv TB of Mycobacterium tuberculosisConfirmatory same project related to Summary assay
492998A Cell Based Secondary Assay to Explore Cytotoxicity in Vero E6 Cells of Compounds that Modulate Non-Replicating, Drug-tolerant Mycobacterium tuberculosisConfirmatory same project related to Summary assay
651617Counterscreen for inhibitors of non replicating M. tb using log phase replicating mycobacteria Measured in Microorganism System Using Plate Reader - 2157-02_Inhibitor_Dose_DryPowder_ActivityConfirmatory same project related to Summary assay
651618Counterscreen for inhibitors of non replicating M. tb using log phase replicating mycobacteria Measured in Microorganism System Using Plate Reader - 2157-02_Inhibitor_Dose_DryPowder_Activity_Set2Confirmatory same project related to Summary assay
651619Secondary assay to identify inhibitors of non-replicating M. tb using luciferase expression without log phase outgrowth Measured in Microorganism System Using Plate Reader - 2157-04_Inhibitor_Dose_DryPowder_Activity_Set2Confirmatory same project related to Summary assay
651620Secondary assay to identify inhibitors of non-replicating M. tb using luciferase expression without log phase outgrowth Measured in Microorganism System Using Plate Reader - 2157-04_Inhibitor_Dose_DryPowder_ActivityConfirmatory same project related to Summary assay
651851Counterscreen for inhibitors of non replicating M. tb using log phase replicating mycobacteriaConfirmatory same project related to Summary assay
651856Secondary assay to identify inhibitors of non-replicating M. tb using luciferase expression without log phase outgrowthConfirmatory same project related to Summary assay
651946HeLa Cytotoxicity Assay Measured in Cell-Based System Using Plate Reader - 7071-04_Other_Dose_DryPowder_ActivityConfirmatory same project related to Summary assay
651949HEK293 Cytotoxicity Assay Measured in Cell-Based System Using Plate Reader - 7071-01_Other_Dose_DryPowder_Activity_Set6Confirmatory same project related to Summary assay
Description:
Keywords: Cytotoxicity Assay, cytotoxicity panel, HepG2

Assay Overview: The Counter Screen use wild-type HepG2 cells from ATCC to counter screen for cytotoxicity at the same concentrations used to confirm compound activity in the primary screen.

Expected Outcome: Compounds identified as hits will be toxic to cells at a compound concentration less than 10 uM. Activity in the assay leads to a reduction in cellular ATP levels which correlates with a decreased luminescence signal from the read reagent (CellTiter-Glo) and indicates cytotoxicity. Compounds that exhibit no cytotoxicity at =<30 uM will be prioritized for additional studies.
Protocol
Mammalian Cytotoxicity Assay Panel SOP
Goal: Standardize and consolidate work efforts of cytotoxicity testing for all probe projects at the Broad. All compounds will be tested in 2 different cell lines for 48 hours and must pass in all to be considered non-toxic. The panel of cell lines will be performed the first and third week of every month.
Assay Protocol:
Day 0, HepG2 cells grown in Triple flask (NUNC) to ~95% confluence (TrypLE Phenol Red free) and resuspended for dispensing at 125,000 cells/mL of DMEM, 10% FBS/Pen/Strep/L-Glutamine (Compact SelecT).
Day 1: Plate cells @5000 per well in 40 microL media (DMEM/10% FBS/Pen/Strep/L-Glutamine) using Corning 8867BC 384 well plates; incubate in standard TC conditions (5% CO2; 95% humidity, 37 degrees C) for 24 hours (Compact SelecT).
Day 2: Add 100 nL compound per well at dose into 40 uL assay volume using a pin tool (CyBi Well). Pin 100 nL cytotoxic compounds, MG132 (CID: 462382) to positive control wells to a final concentration of 16 microM (100 nL 6.4 mM DMSO stock). Incubate for 48 hours at 37 degrees C in Liconic incubator, 95% humidity 5% CO2.
Day 4: Remove plate from incubator to cool for 15 minutes to room temperature; add 20 microL 50% Promega CellTiter-Glo (diluted 1:1 with PBS, pH 7.4) with Thermo Combi.
Incubate at RT for 5 minutes.
Read on Perkin-Elmer EnVision with US LUM settings for 0.1 sec per well.
Comment
PRESENCE OF CONTROLS: Neutral control wells (NC; n=28) and positive control wells (PC; n=8) were included on every plate.
EXPECTED OUTCOME: Active compounds result in decreasing readout signal.
ACTIVE CONCENTRATION LIMIT:
For each sample, the highest valid tested concentration (Max_Concentration) was determined and the active concentration limit (AC_limit) was set to equal (2)(Max_Concentration).
NORMALIZATION:
The raw signals of the plate wells were normalized using the 'Neutral Controls Minus Inhibitors' method in Genedata Assay Analyzer (v10.0.2):
The median raw signal of the intraplate neutral control wells was set to a normalized activity value of 0.
The median raw signal of the intraplate positive control wells was set to a normalized activity value of -100.
Experimental wells values were scaled to this range.
PATTERN CORRECTION: No plate pattern correction algorithm from Genedata Condoseo (v.10.0.2) was applied.
MEASUREMENT USED TO DETERMINE ACTIVE CONCENTRATION (AC): AC50
AC values were calculated using the curve fitting strategies in Genedata Screener Condoseo (7.0.3).
AC values were calculated up to the active concentration limit described for each sample.
pAC was set to equal -1*log10(AC)
PUBCHEM_ACTIVITY_OUTCOME:
Activity_Outcome = 1 (inactive) when:
a) compound shows activity but in a direction opposite to the expected outcome
in these cases, values describing curve fitting parameters (Sinf, S0, Hill Slope, log_AC50, log_AC50_SE) are set to null
b) curve fit is constant where activity is > -30% and < 30% at all tested concentrations, or
c) AC > AC_limit
Activity_Outcome = 2 (active) when:
AC <= AC_limit
Activity_Outcome = 3 (inconclusive) when:
a) Curve fitting strategy resulted in a constant fit with activity >= -70% but <= -30%, or
b) The fit was deemed not valid due to poor fit quality.
PUBCHEM_ACTIVITY_SCORE:
If PUBCHEM_ACTIVITY_OUTCOME = 1 (inactive) or 3 (inconclusive),
then PUBCHEM_ACTIVITY_SCORE = 0
If PUBCHEM_ACTIVITY_OUTCOME = 2 (active)
then PUBCHEM_ACTIVITY_SCORE = (10)(pAC)
Scores relate to AC in this manner:
120 = 1 pM
90 = 1 nM
60 = 1 uM
30 = 1 mM
0 = 1 M
When the active concentration (AC) is calculated to be greater than the highest valid tested concentration (Max_Concentration), the PUBCHEM_ACTIVITY_SCORE is calculated using Max_Concentration as the basis.
When the active concentration (AC) is calculated to be less than the lowest tested concentration, the PUBCHEM_ACTIVITY_SCORE is calculated using the lowest tested concentration as the basis.
Note:
The individual dose data point columns ('Activity_at_xxuM') reported here represent the median of valid (unmasked) replicate observations at each concentration. These values are the inputs to a curve fitting algorithm.
All other data columns represent values which are derived during the curve fitting algorithm; this may sometimes include automatic further masking of some replicate data points.
Occasionally this results in perceived inconsistencies: for example, between the derived 'Maximal_Activity' and the apparent most active data point.
Categorized Comment - additional comments and annotations
From PubChem:
Assay Format: Cell-based
Assay Type: Toxicity
Assay Cell Type: HEPG2
Result Definitions
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TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1AC50_Qualifier'>','=', or '<'String
2AC50_uM*The concentration at which activity reaches 50% of the maximumFloatμM
3pAC50_MEqual to -1*log10(AC50)String
4Hill_SlopeThe slope at AC50Float
5S0_(%)The fitted activity value at zero concentrationFloat%
6Sinf_(%)The fitted activity value at infinite concentrationFloat%
7Num_PointsThe number of data points used to generate the plotInteger
8Max_Activity_(%)The maximum activity value observed, based on mean of replicates per concentrationFloat%
9Max_Activity_Conc_uMThe concentration at which the maximum activity is observedFloatμM
10Max_Concentration_uMMaximum valid test concentrationFloatμM
11Activity_at_0.195uM_(%) (0.195μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
12Activity_at_0.38uM_(%) (0.38μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
13Activity_at_0.8uM_(%) (0.8μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
14Activity_at_1uM_(%) (1μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
15Activity_at_1.6uM_(%) (1.6μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
16Activity_at_1.95uM_(%) (1.95μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
17Activity_at_3uM_(%) (3μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
18Activity_at_3.8uM_(%) (3.8μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
19Activity_at_6uM_(%) (6μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
20Activity_at_8uM_(%) (8μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
21Activity_at_12uM_(%) (12μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
22Activity_at_16uM_(%) (16μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
23Activity_at_26uM_(%) (26μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
24Activity_at_30uM_(%) (30μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
25Activity_at_60uM_(%) (60μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
26Activity_at_120uM_(%) (120μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%

* Activity Concentration. ** Test Concentration.
Additional Information
Grant Number: 1 R03 MH087444-01

Data Table (Concise)
Data Table ( Complete ):     View Active Data    View All Data
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